The overall objective of the proposed research is to investigate the biochemistry and biology of the tissue-specific isozymes of human delta- aminolevulinate synthase (ALA-S), the first enzyme and the rate-limiting step of heme biosynthesis. These studies of the housekeeping isozyme (ALAS1) and the erythroid-specific isozyme (ALAS2) are designed to provide fundamental information and understanding of 1) the physicokinetic characteristics of the tissue-specific human ALA-S isozymes, 2) the nature of the normal processing and maturation of the ALA-s isozymes during their transport and incorporation into mitochondria, 3) the genetic and phenotypic heterogeneity underlying the congenital hematological disorder, X-linked sideorblastic anemia (XLSA), 4) the biochemical and cell biologic phenotype resulting from ALAS1 deficiency and 5) the transcriptional regulation of ALAS1 by heme in HepG2 cells. Our isolation and sequencing of the full-length human cDNAs and genomic sequences encoding ALAS1 and ALAS2, expression of both isozymes in prokaryotic and eukaryotic systems, and discovery that mutations in ALAS2 cause XLSA provide the unique and necessary resources to accomplish these goals. The cellular processing and mitochondrial targeting of these isozymes will be studied in a variety of cells including COS-1. CHO, HePG2, and K562 to determine the cleavage site of the mitochondrial import leader sequence and the nature of any further mitochondrial proteolytic processing. The mature mitochondrial forms of both isozymes will be characterized including pH optima, K, K pyridoxal 5'-phosphate (PLP) binding, pI, stability, molecular weight and shape, and effects of metals, ions and nucleotide. Purified recombinant ALAS1 and ALAS2 will be used for antibody production and for crystallization and X-ray diffraction to determine their three-dimensional structures. Site-directed chemical modification and site-directed mutagenesis will be used to identify and evaluate residues involved in the active site of both isozymes, to identify the PLP co-factor binding site, to characterize the motifs implicated in heme binding, to determine the residues involved in proteolytic processing and degradation and to detect sites involved in subunit association. A cellular model for non- erythroid heme deficiency will be produced by introduction of specific ALAS1 mutations into murine embryonic stem cells and human HepG2 cells by homologous recombination. Such heme synthesis-deficient cells could provide valuable model systems for investigations of various heme- dependent systems such as generalized cytochrome deficiencies and the resulting biochemical phenotypes may suggest analogous human genetic disorders. Efforts will be directed to determine the molecular events mediating the repression of ALAS1 transcription by heme. A sensitive and accurate RT-PCR method for the quantitation of ALAS1 mRNA will be used to evaluate the heme-mediated control of ALAS1 transcription in HepG2 cells. The transcriptionally active regions of the ALAS1 gene will be identified by DNase I mapping and footprinting and the transcription factor(s) interacting with these in response to heme will be characterized.